Abstract: Nanoparticles have important biomedical applications ranging from the treatment of human disease with gene therapy to understanding basic cellular functions with fluorescent probes. It is now possible to synthesize nanoparticles with nearly any property or size, direct them to specific cells, and functionalize them to target intracellular locations, but delivering nanoparticles across the plasma membrane to reach their targets remains a challenge. We have recently demonstrated the first method for non-invasive delivery of semiconductor nanoparticles, known as quantum dots (QDs), to the cytosol of multiple cells simultaneously. Delivery requires pyrenebutyrate in combination with a cationic peptide for direct interaction of the QD with the plasma membrane. The ability of QDs to cross the plasma membrane offers exciting possibilities for the delivery of other nanoparticles to living cells. The first goal of this research program is to determine the molecular and cellular mechanism of pyrenebutyrate-mediated delivery and extend it to other nanoparticles with the ultimate goal of targeting specific intracellular sites with nanoparticles of choice. Novel imaging methods, including single particle tracking fluorescence microscopy, will be used to probe the motion of the nanoparticle and its interaction with pyrenebutyrate as it moves across the plasma membrane and through the cytosol. As pyrenebutyrate-mediated delivery may not be suitable for all nanoparticles and all applications, we will also work to develop a suite of cytosolic delivery and targeting methods that are based on the well-characterized endosomal uptake pathways. Both delivery methods will be carried out in conjunction with studies of cellular response to nanoparticles that aim to optimize delivery and minimize disruption. In the course of this research, QDs, which are sufficiently bright for imaging at the single particle level, will be used to probe fundamental questions of cellular transport including diffusion through the crowded cellular environment, vesicle-mediated transport, and nuclear targeting. Public Health Relevance: This research addresses a fundamental challenge in the use of nanotechnology for the benefit of human health;the delivery of nanoparticles into living cells. Two delivery methods, translocation and endocytosis, will be explored in conjunction with studies of cellular response for optimal delivery and minimal disruption. These studies will use a modular scheme for functionalization that can be extended to a wide variety of nanoparticles for broad impact in many different disciplines including cancer therapy, gene delivery, and cellular imaging.